Bioprocessing Basics JUNE 2020 Bioreactors and Fermenters: Powerful Tools for Resolving Cultivation Bottlenecks Ulrike Rasche Advanced Technologies Facilitate Scale-up and Technology Transfer Cynthia Challener A Beginner’s Guide to Bioprocess Modes: Batch, Fed-Batch, and Continuous Fermentation Ying Yang and Ma Sha Sponsored by This custom ebook is sponsored by Eppendorf and presented in partnership with BioPharm International. Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks SPONSORED CONTENT APPLICATION NOTE Bioreactors and Fermenters: Powerful Tools for Resolving Cultivation Bottlenecks Ulrike Rasche Shake flasks, cell culture dishes, and Introduction T-flasks are the first vessels that come Many applications are well served by the to mind when we think about cultivation cultivation of bacteria or yeast in shake systems for growing eukaryotic and flasks and cells in dishes or T-flasks. prokaryotic cells in the lab. Bioreactors Bioreactors and fermenters, however, improve productivity and save work, and fermenters are another alternative time, and lab space for scientists, who: to consider if we need larger quantities • need large quantities of cells, of cells, increased efficiency of microbes, or the products cultivation, or enhanced reproducibility. they express In this white paper, we explain the key • would like to improve the characteristics of stirred-tank bioreactors reproducibility of growth, the product and which organisms are typically grown formation, or the product quality in them. Using specific examples, • would like to systematically we demonstrate how bioreactors compare different growth conditions and fermenters can help to resolve • would like to increase the cultivation bottlenecks. cultivation efficiency 2 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks What are bioreactors Stirred-tank bioreactors and fermenters? Though many types of bioreactors exist, Broadly speaking, bioreactors and we will focus on stirred-tank bioreactors. fermenters are culture systems to The name is accurately descriptive. produce cells or organisms. They Cultivation takes place in the bioreactor are used in various applications, tank—often called a vessel—and the including basic research and culture is mixed by stirring (instead development, and the manufacturing of shaking, for example). Stirred-tank of biopharmaceuticals, food and bioreactors come in different sizes (for food additives, chemicals, and other cultures of a few milliliters to thousands of products. A broad range of cell types liters) and are made of various materials and organisms can be cultivated in (usually glass, plastic or stainless steel). bioreactors and fermenters, including The basic components and functioning cells (like mammalian cell lines, insect of stirred-tank bioreactors are always the cells, and stem cells), microorganisms same. A stirred-tank bioreactor system (like bacteria, yeasts, and fungi), as well consists of several parts (Figure 1): as plant cells and algae. • A vessel that is filled with the Bioreactor and fermenter are two words medium in which cells are cultivated for basically the same thing. Scientists • A head plate to close the vessel who cultivate bacteria, yeast, or fungi often use the term fermenter. The • Components within or attached term bioreactor to the vessel or the head plate to often relates to Figure 1: Stirred-tank bioreactor system consisting of bio- the cultivation process control station, vessel, and bioprocess control soft- of mammalian ware. The BioFlo 120 bioprocess control station is shown cells, but is also generically used. When we talk about bioreactors in this white paper, we usually mean systems for the cultivation of microbes or mammalian cells. 3 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks measure and adjust the culturing oxygen is transferred from the surrounding conditions, such as feed lines air to the culture medium. This process and sensors is more efficient in shake flasks than in • A control system comprising static cultures because shaking increases external components used to exposure of the liquid surface. In adjust the culturing conditions (e.g., bioreactors, air or pure oxygen (coming for pumps) and control software example from a compressed air cylinder) is usually introduced to the culture. Creating optimal cultivation conditions With the use of spargers, the gas/liquid Like incubators and shakers, bioreactors interface can be increased and the oxygen allow for the creation of optimal supply maximized. Oxygen is important environmental conditions for the for culture growth, and the amount of growth of cells or microbes. They differ, oxygen dissolved in the medium (dissolved however, in how these are established. oxygen concentration, DO) is continuously Culture mixing. Instead of mixing by measured with a DO sensor. shaking, in a bioreactor, the culture is To keep DO at setpoint, a DO cascade stirred with an impeller. The impeller is is often set up in and executed by the mounted to the impeller shaft, which bioprocess control software. Figure 2 in turn is connected to a motor. In a shows an example for a typical DO bioreactor, not only are bacterial, yeast, cascade. If DO drops below setpoint, first and suspension cell cultures constantly the agitation speed is gradually increased mixed, but so too are the cultures of up to 1,200 rpm to increase oxygen adherent cells attached to a growth matrix. transfer from the surrounding air. If this Tempering. To obtain the right cultivation temperature, a bioreactor does not Figure 2: Dissolved oxygen cascade need to be placed inside a shaker or incubator but can remain on the lab bench. The temperature of the culture medium is continuously monitored with a temperature sensor. To regulate it, the vessel is placed in a thermowell, wrapped with a heating blanket or has a water jacket. Cooling is possible as well. Establishing aerobic or anaerobic conditions. In a shaker or incubator, 4 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks is not sufficient to keep DO at setpoint, incubator atmosphere is measured and the gas flow rate is increased up to three controlled, rather than the medium pH. standard liters per minute (SLPM). As a Bioreactors also differ in that a basic final measure, the oxygen concentration solution is often added to compensate in the gas mix is increased, shifting from for acidification during culture growth. gassing with air (containing 21% oxygen) For microbial cultures in bioreactors, toward gassing with pure oxygen. This basic and acid solutions are commonly is just an example. The minimum and used for pH adjustment. This is different maximum values of agitation, gas flow from cultures in shake flasks, where the rate, and oxygen concentration can be culture pH is usually not controlled. optimized depending on the organism Control of parameters at setpoint. In and process needs. bioreactors, different components and Anaerobic conditions can be established by the control software play together to gassing with N2 or other anaerobic gases. control pH, temperature, and dissolved oxygen at the desired setpoint. The pH control. To regulate the pH of parameters are constantly measured carbonate-buffered cell culture media, using pH, temperature, and DO sensors. cell cultures in flasks or dishes are The sensors transmit the information to usually placed in CO incubators. In 2 the bioprocess control software, which bioreactors, the principle is the same; regulates the addition of CO and liquid CO is introduced to the culture from a 2 2 pH agents, the activity of tempering compressed gas cylinder. In bioreactors, devices, agitation, and the gassing with the medium pH is continuously air and/or O2. measured using a pH sensor and CO2 is added as needed. This is different from A typical bioprocess run the situation in a CO incubator, in which 2 To maintain cultures, scientists usually the CO concentration in the internal 2 keep them in cultivation systems within incubators. Bioreactors are usually used Sponsor’s Content for a specific experiment or production run, which may last hours, days, or BioProcessing weeks, depending on the Introduction organism and application. A bioprocess run typically comprises the following steps (Figure 3): 5 JUNE 2020 | BIOPHARM INTERNATIONAL SPONSORED CONTENT Bioreactors and Fermenters: Powerful Tools for Bioreactors Scale Up, Fermentation and Fermenters Tech Transfer Resolving Cultivation Bottlenecks 1. Preculture: The medium in the process parameter setpoints in the bioreactor is inoculated with a bioprocess control software. preculture. Often, the preculture 3. Inoculation: Once the bioreactor is is grown in a shaker or incubator. prepared, the medium is inoculated. Sometimes, smaller bioreactors are 4. Cultivation period: During the used to grow precultures for the cultivation period, agitation, pH, inoculation of larger bioreactors. temperature, and DO are typically 2. Bioreactor preparation: The monitored and controlled in real bioreactor is prepared in parallel